CN113388666A - D-A-D type FRET DNA nano machine and preparation method and application thereof - Google Patents
D-A-D type FRET DNA nano machine and preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to the technical field of miRNA detection, in particular to a D-A-D type FRET DNA nano machine and a preparation method and application thereof, wherein four annealed hairpin DNAs are used for sequentially modifying a DNA tetrahedron to obtain the D-A-D type FRET DNA nano machine, and the four hairpin DNAs are HMg, H1-FAM, H2-TAMRA and H3-FAM respectively. According to the invention, based on HMg shearing and catalytic hairpin amplification, a D-A-D type FRET DNA nano machine is constructed, the nano machine can realize high-sensitivity detection and in-situ fluorescence imaging of miRNA, the fluorescence FRET efficiency is enhanced, the detection sensitivity is improved, and the technical problems of poor cell membrane penetrability and poor biological stability of hairpin DNA are solved.
Description
Technical Field
The invention relates to the technical field of miRNA detection, in particular to a D-A-D type FRET DNA nano machine and a preparation method and application thereof.
Background
The early discovery of malignant tumors has great significance for preventing and treating cancers, but because the concentration of tumor markers in bodies of early cancer patients is low or the number of tumor cells is small, the conventional method is difficult to accurately detect, and the important significance is brought to the construction of an integrated diagnosis and treatment platform by searching for representative tumor markers, searching for high-sensitivity detection means and constructing a new cyclic amplification probe.
mirnas are single-stranded, small, non-coding RNAs that play important roles in a variety of cellular processes, such as cell proliferation, tumorigenesis, and embryonic development. Research shows that the expression disorder of miRNA is related to various human diseases, especially cancer, and is related to the over-expression of various miRNA. Therefore, mirnas can be important markers for early diagnosis of cancer. The fluorescence analysis method can realize the analysis of low-concentration miRNA in early cancer cells, is favored by analysts with the advantages of high sensitivity, good selectivity, simple operation and the like, and is widely applied to the fields of biochemical analysis, drug detection and the like.
Hairpin DNA is often used as a fluorescent probe for intracellular detection, but is often limited due to poor cell membrane penetration and biological stability. In view of the above, there is a need to provide a nanomachine with better cell membrane permeability and biostability to achieve more efficient live cell imaging.
Disclosure of Invention
The invention provides a D-A-D type FRET DNA nano machine and a preparation method and application thereof, aiming at the technical problems of poor cell membrane penetrability and poor biological stability of hairpin DNA, and the D-A-D type FRET DNA nano machine is constructed based on HMg shearing and Catalytic Hairpin (CHA) amplification, and can realize high-sensitivity detection and in-situ fluorescence imaging of miRNA.
In a first aspect, the invention provides a method for preparing a D-A-D FRET DNA nano-machine, wherein four pieces of annealed hairpin DNA are used for sequentially modifying a DNA tetrahedron to obtain the D-A-D FRET DNA nano-machine, and the four pieces of hairpin DNA are HMg (Mg) respectively2+DNAzyme), H1-FAM, H2-TAMRA and H3-FAM, the sequence of HMg is shown in SEQ ID No.5, the sequence of H1-FAM is shown in SEQ ID No.6, and the sequence of H2-TAMRAIs shown as SEQ ID No.7, and the sequence of H3-FAM is shown as SEQ ID No. 8.
Further, the method comprises the following steps:
(1) diluting the four hairpin DNAs with Tris-HCl buffer solutions respectively and annealing;
(2) then the four annealed hairpin DNAs are mixed with DNA tetrahedron proportion in sequence and reacted at 37 ℃, and the molar ratio of the four hairpin DNAs to the DNA tetrahedron is 1:1:1:1: 1.
further, the step (1) is specifically as follows:
and (3) respectively diluting the four hairpin DNAs into 1 mu M dispersions by using Tris-HCl buffer solution, respectively heating the four dispersions at 95 ℃ for 5min, and naturally cooling to room temperature to finish annealing treatment.
Further, the concentration of the Tris-HCl buffer solution was 20mM, and the pH was 7.4.
Further, the DNA tetrahedron is prepared according to the following method:
sequentially adding four long oligonucleotide chains into a TM buffer solution to obtain a uniform mixture, heating and immediately cooling to obtain a DNA tetrahedron, wherein the four long oligonucleotide chains are respectively TDNa, TDNb, TDNc and TDNd, the sequence of TDNa is shown in SEQ ID No.1, the sequence of TDNb is shown in SEQ ID No.2, the sequence of TDNc is shown in SEQ ID No.3, and the sequence of TDNd is shown in SEQ ID No. 4.
Further, the heating temperature of the mixture is 95 ℃ and the heating time is 5 min; the cooling temperature is 4 deg.C, and the cooling time is 30 min.
Further, the concentrations of TDNa, TDNb, TDNc and TDNd in the DNA tetrahedron are all 1 μ M.
Further, the concentration of Tris-HCl in the TM buffer solution is 20mM, MgCl2Has a concentration of 50mM, and the pH of the TM buffer solution is 8.0.
In a second aspect, the invention provides a D-A-D type FRET DNA nano-machine prepared by the preparation method.
In a third aspect, the invention provides an application of the D-A-D type FRET DNA nano-machine in miRNA detection and/or imaging in cells.
Advantageous effects
The invention adopts DNAzyme shearing and CHA (catalysis hairpin) double DNA circulation amplification, takes a DNA tetrahedron as a core, and carries out specificity modification through four vertexes of the DNA tetrahedron to ensure that the DNA tetrahedron carries hairpin DNAzyme with the functions of recognition and shearing and three hairpins of Y-type CHA without enzyme catalysis, on one hand, DNAzyme, CHA and FRET (fluorescence resonance energy transfer) are combined, a novel D-A-D (donor-acceptor-donor) FRET mode is constructed, and the FRET efficiency and the detection sensitivity are improved; on the other hand, the hairpin-modified tetrahedron can enter cells through endocytosis, has good cell membrane penetrability and biological stability, and can further complete recognition, shearing and CHA processes in the cells, thereby constructing a cross-linked network structure taking the DNA tetrahedron and the Y structure as nodes and providing a new strategy for realizing biological imaging.
Drawings
In order to more clearly illustrate the embodiments or technical solutions in the prior art of the present invention, the drawings used in the description of the embodiments or prior art will be briefly described below, and it is obvious for those skilled in the art that other drawings can be obtained based on these drawings without creative efforts.
FIG. 1 is a schematic diagram of the assembly and cell imaging of a TDNH nano machine;
FIG. 2 shows the fluorescence emission spectra of HMg-FAM under different conditions;
FIG. 3 is a representation of an ion selectivity assay of HMg;
FIG. 4 is a polyacrylamide electrophoretogram of the HMg shear and CHA process;
FIG. 5 is a graph of fluorescence intensity and a FRET histogram for different Y-FRET encounter modes;
FIG. 6 is an agarose electrophoresis of TDNH;
FIG. 7 is a representation of Y-TDNH;
FIG. 8 is a signal diagram of miRNA-21 (D);
FIG. 9 is a diagram of TDNH selectivity against different miRNA or base-mismatched RNAs;
FIG. 10 is an agarose electrophoresis picture of DNase I degrading TDNH;
FIG. 11 is a bar graph of the results of a CCK-8 cytotoxicity assay;
FIG. 12 is a photograph of fluorescent images of miRNA-21 in HeLa cells by three sets of nanomachines;
FIG. 13 is a DAPI fluorescence image of HMg/H1/H2/H3 and TDNH-treated HeLa cells;
FIG. 14 is a confocal image of HEK293, HeLa, HepG2 and MCF-7 cells.
Detailed Description
In order to make those skilled in the art better understand the technical solution of the present invention, the technical solution in the embodiment of the present invention will be clearly and completely described below with reference to the drawings in the embodiment of the present invention, and it is obvious that the described embodiment is only a part of the embodiment of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
DMEM culture solution, PBS buffer solution (1 x 0.0067M) and trypsin used in the invention are purchased from general electric China medical Co., Ltd; cervical cancer cells (Hela), breast cancer cells (MCF-7), liver cancer cells (HepG 2) and human embryonic kidney cells (HEK 293) were purchased from Beijing Yingjinjing biomedical technologies, Inc.; magnesium chloride hexahydrate available from Shanghai Aladdin Chemicals; the CCK-8 kit and DAPI were purchased from Shanghai Bintian Biotechnology Ltd.
The deionized water used in the invention is prepared by a water purification system with the resistance of 18.2M omega/cm.
The DNA and RNA sequences used in the present invention were purchased from Shanghai Producer, Inc., and are specifically shown in Table 1 below.
TABLE 1 DNA and RNA sequences used in the invention
Name (R) | Sequence (5 '-3') | Description of the invention | SEQ ID No. |
TDNa | TATCACCAGGCAGTTGACAGTGTAGCAAGCTGTAATAGATGCGAGGGTCCAATACTCGCGATTCGGCACA | 1 | |
TDNb | TCAGGGTCTACGGCTTCAACTGCCTGGTGATAAAACGACACTACGTGGGAATCTACTATGGCGGCTCTTC | 2 | |
TDNc | GCTCACTCTTCCACTTTTCAGACTTAGGAATGTGCTTCCCACGTAGTGTCGTTTGTATTGGACCCTCGCAT | 3 | |
TDNd | TCAATGTCATCGGATACATTCCTAAGTCTGAAACATTACAGCTTGCTACACGAGAAGAGCCGCCATAGTA | 4 | |
HMg | CAAAAAGAGAGCAACATCAGTCCTrAGTCTTTTTTTTTGATCCGAGCCGGACGAAGGGATAAGCTATGTGCCGAATCGCGA | 5 | |
HMg-FAM | CAAAAAGAGAGCAACATCAGTCCTrAGTCTTTTTTTTTGATCCGAGCCGGACGAAGGGATAAGCTATGTGCCGAATCGCGA | 5’- |
5 |
H1 | AGCCGTAGACCCTGAGAGCAACATCAGTCCTGTATAGCCCAAGAGAGTAAGGACTGATGTTGCTCTCTTTTTG | 6 | |
H1-FAM | AGCCGTAGACCCTGAGAGCAACATCAGTCCTGTATAGCCCAAGAGAGTAAGGACTGATGTTGCTCTCTTTTTG | T’- |
6 |
H2 | AGTGGAAGAGTGAGCGTATAGCCCAAGAGAGCACCTACGCAAAAAGATACTCTCTTGGGCTATACAGGACTGA | 7 | |
H2-TAMRA | AGTGGAAGAGTGAGCGTATAGCCCAAGAGAGCACCTACGCAAAAAGATACTCTCTTGGGCTATACAGGACTGA | T’- |
7 |
H3 | ATCCGATGACATTGACACCTACGCAAAAAGAGAGCAACATCAGTCCTTATCTTTTTGCGTAGGTGCTCTCTTG | 8 | |
H3-FAM | ATCCGATGACATTGACACCTACGCAAAAAGAGAGCAACATCAGTCCTTATCTTTTTGCGTAGGTGCTCTCTTG | T’- |
8 |
H3-TAMRA | ATCCGATGACATTGACACCTACGCAAAAAGAGAGCAACATCAGTCCTTATCTTTTTGCGTAGGTGCTCTCTTG | T’- |
8 |
DNA1 | CAAAAAGAGAGCAACATCAGTCCT | 9 | |
miRNA-21 | |
10 | |
miRNA-21(D) | TAGCTTATCAGACTGATGTTGA | 11 | |
miRNA-21(D)-BHQ1 | TAGCTTATCAGACTGATGTTGA | 3’-BHQ1 | 11 |
miRNA-16(D) | TAGCAGCACGTAAATATTGGCG | 12 | |
miRNA-26a(D) | TTCAAGTAATCCAGGATAGGCT | 13 | |
miRNA-141(D) | TAACACTGTCTGGTAAAGATGG | 14 | |
one-base mismatch T1 | TAGCTTATCAGACTAATGTTGA | 15 | |
two-base mismatch T2 | TAGCTTATCAGACTCATCTTGA | 16 | |
three-base mismatch T3 | TAGCTTTTCAGACTCAAGTTGA | 17 |
Example 1
The four long oligonucleotides TDNa, TDNb, TDNc and TDNd are added into TM buffer solution (20 mM Tris-HCl, 50mM MgCl)2pH8.0) to obtain a mixture, heating the mixture at 95 ℃ for 5min, then immediately cooling the mixture at 4 ℃ for 30min to finally obtain a DNA Tetrahedron (TDNs) with the concentration of 1 mu M, and storing the DNA tetrahedron at 4 ℃ for later use.
The four hairpin DNAs of HMg, H1-FAM, H2-TAMRA and H3-FAM are respectively diluted by Tris-HCl buffer solution (20 mM, pH7.4) and annealed, and the annealing process is as follows: heating at 95 ℃ for 5min, naturally cooling to room temperature, mixing the four annealed hairpin DNAs and synthesized TDNs in a ratio of 1:1:1:1:1, and reacting for 2h under a water bath condition at 37 ℃ to obtain a hairpin-modified functionalized DNA Tetrahedron (TDNH), namely a D-A-D type FRET DNA nano-machine, wherein the TDNH can be used for detecting miRNA-21 in cells.
And incubating TDNH by taking 100 mu L of miRNA-21 (D) with the concentration of 0.1 mu M as an initiator at the incubation temperature of 37 ℃ for 4h to obtain Y-TDNH.
The principle of detecting miRNA-21 in cells by TDNH is as follows:
as shown in figure 1A, the invention designs a hairpin HMg capable of specifically recognizing miRNA21, under the condition that miRNA-21 and Mg2+ exist at the same time, miRNA-21 recognizes HMg to trigger hairpin configuration change, and then specific site shearing is carried out under the action of Mg2 +. Because of the reduction of base complementation, the HMg subjected to self-shearing can be automatically separated from miRNA-21, and simultaneously generate short-chain DNA1, miRNA21 can be combined with hairpin HMg again to form cycle1 (cycle 1), so that the relative concentration of miRNA21 is increased, and the detection sensitivity is improved; meanwhile, the short-chain DNA1 can be used as a priming chain to participate in CHA reactions of hairpin H1-FAM, H2-TAMRA and H3-FAM without enzyme catalysis based on the theory of foothold mediation. Firstly, DNA1 opens the hairpin H1, and at the same time the naked end of H1 opens the hairpin H2, H2 opens H3, then the naked end of H3 replaces DNA1 base-complementary paired with H1, and further DNA1 opens more H1, H2, H3, constituting cycle2 (cycle 2). Therefore, a stable H1-H2-H3Y-type DNA structure is formed, and simultaneously, three fluorescent groups meet at the center of the Y-type structure to draw the distance between fluorophores to generate D-A-D Y type fluorescence resonance energy transfer, and the detection sensitivity is further improved through a ratio signal.
Based on the design thought, TDNs are used as carriers, and hairpins HMg, H1, H2 and H3 are respectively introduced into the carriers according to the base complementary pairing principle of four vertex extension ends of the carriers. As shown in FIG. 1B, TDNH can enter cells by endocytosis and encounter miRNA-21 and Mg in the cells2+The same shear effect caused the conformational change, releasing DNA1 which opened H1 on the first TDNH, and thus H2 on the adjacent second TDNH and H3 on the third TDNH. The three TDNH take the formed Y structure as a connection point and extend to four sides in an emission shape to form a stable Y-shaped network structure (Y-TDNH). Furthermore, the three-dimensional structure of TDNH allows TDNH-mediated Catalytic Hairpin (CHA) reactions to extend in different directions, facilitating the capture of free reactant TDNH from the surrounding environment. More importantly, once the hairpin at one TDNH vertex participates in the CHA reaction, the hairpins at the other three vertices are necessarily brought into a DNA network structure, no matter the hairpins are sheared or CHA, the local concentration of reactants is greatly increased, the collision chance, multiple reaction directions and the synergistic effect of increasing the local reactant concentration are further increased, the reaction rate is necessarily greatly increased, and the fluorescence detection of miRNA-21 and the in-situ cell imaging in cells are facilitated. From the construction of the DNA nano machine TDNH to the formation of the Y-TDNH network, an important method strategy is provided for realizing rapid and accurate cancer diagnosis.
Test example 1 in vitro fluorescence and electrophoresis experiments
In vitro experiments were performed using miRNA-21 (D) sequences identical to miRNA-21.
First, the specific recognition of self-cleaving hairpin HMg and miRNA-21 and the recognition of Mg by DNAzyme were verified2+Dependence of (2). FAM and BHQ1 fluorophores were labeled at the 5 'end of HMg and the 3' end of miRNA-21 (D) using fluorescent labeling technique, and 100nM HMg and miRNA-21 (D) -BHQ1 solutions were prepared using Tris-HCl buffer (20 mM, pH 7.4) and heated at 37 ℃ for 2 h. Separately detect HMg-FAM + miRNA-21 (D) -BHQ1, HMg-FAM + miRNA-21 (D) -BHQ1+ Mg2+And HMg-fluorescence emission pattern in the case of FAM.
The results are shown in FIG. 2, curves a, b and c represent HMg-FAM + miRNA-21 (D) -BHQ1, HMg-FAM + miRNA-21 (D) -BHQ1+ Mg, respectively2+And HMg-FAM, which when HMg-FAM binds to both miRNA-21 (D) -BHQ1, causes a conformational change in HMg, bringing FAM into proximity with BHQ1 fluorophore, resulting in fluorescence quenching; presence of Mg2+When it is Mg2+Cleavage of the hairpin at a particular position is triggered to effect recovery of fluorescence.
Next, to evaluate DNAzyme for Mg2+Has good selectivity, takes HMg-FAM and DNA21-BHQ1 as recognition quenching as reaction substrates, adds different metal ions with the concentration of 25mM for replacement, and uses an F-4600 fluorescence spectrophotometer to measure the fluorescence intensity of the sample (E)x/Em=488/520 nm). As shown in FIG. 3, only Mg2+DNAzymes have only a strong shearing function when present.
To verify the stability of the four hairpins after annealing and the occurrence of DNAzyme cleavage and CHA amplification, 12% polyacrylamide electrophoresis was performed. A solution of HMg, a solution of H1, a solution of H2, a solution of H3 and a solution of miRNA-21 (D) were prepared at a concentration of 1. mu.M using Tris-HCl buffer (20 mM, pH 7.4), and heated at 37 ℃ for 2 hours. Then, the obtained sample, miRNA-21 (D) + HMG sample, miRNA-21 (D) + HMg + H1+ H2+ H3 sample and miRNA-21 (D) + HMg + Mg sample are mixed2+And sample miRNA-21 (D) + HMg + H1+ H2+ H3+ Mg2+The imaging analysis was performed using an IR-980A biophoresis image analysis system, run on a 135V PAGE gel electrophoresis apparatus for 1.5 h.
In FIG. 4, the 1-5 bands are miRNA-21 (D), HMg, H1, H2 and H3 in sequence, and it can be seen that the annealed hairpin DNA has separate and clear marks; by comparing strips 6, 8, in the absence of Mg2+Under the conditions ofHMg binding to miRNA-21 (D) results in increased electrophoretic mobility, an upward shift in position compared to the HMg band, and conversely an increase in Mg2+In the presence of the miRNA-21 (D), HMg and the miRNA-21 (D) are sheared, a HMg chain is shortened, the electrophoretic mobility is accelerated, a strip mark is moved downwards, and meanwhile a cut DNA1 chain is combined with the miRNA-21 (D) to form a new mark; by comparing bands 7 and 9, mixing miRNA-21 (D) with HMg, H1, H2, and H3 shears and initiates CHA, resulting in a reduced electrophoretic mobility of the Y structure, with the band moving upward.
The results of test example 1 indicate that the combination of DNAzyme and CHA is feasible.
Test example 2 experiment of fluorescence conversion efficiency
In order to investigate the fluorescence conversion efficiency under different fluorescent labels, sheared strand DNA1 ( DNA 1, 50 nM) is added into solutions of H1, H2 and H3 (100 nM, 100 uL) which are annealed and different fluorescent labels as a priming strand, the solutions are reacted for 2H at room temperature, DNA1 catalyzes a hairpin to form a Y structure, fluorescent groups meet in the middle of Y and FRET occurs, five meeting modes are set, namely a double donor D-D (DNA 1+ H1-FAM + H2-FAM + H3), a single donor D (DNA 1+ H1-FAM + H2+ H3), a donor-acceptor D-A (DNA 1+ H1-FAM + H2-TAMRA + H3), a single donor-double acceptor D-A (DNA 1+ H1-FAM + H2-TAA + MRH 3-TAMRA + MRA-A-3) and a donor-double acceptor D-DNA 8672 (DNA 8672 + TAM-3), the samples were then fluorescence detected and the fluorescence at 520nm was normalized for comparison of FRTE efficiency. As shown in FIGS. 5A and 5B, curves a to e show the changes in fluorescence intensity of FAM in D-D, D, D-A, D-A-A, D-A-D single comparison, respectively, with the FAM in D-A-D mode decreasing most in fluorescence intensity and being quenched most effectively; simultaneously with D-A (F)A/FD=0.58)、D-A-A(FA/FD= 0.69) comparison, D-A-D (F)A/FD= 1.25) has higher FRET efficiency.
Test example 3 characterization of TDNH and Y-TDNH
Because TDNH has larger molecular weight, agarose electrophoresis capable of distinguishing biological macromolecules is selected to characterize the assembly and reaction process of Y-TDNH. TDNs, TDNH and Y-TDNH of example 1 were characterized by electrophoresis on a 1% agarose gel (80V, 30 min) at room temperature, and the gel was stained with Ethidium Bromide (EB) prior to gel formation and analyzed by imaging with a gel imaging system. As shown in FIG. 6, lanes 1-4, the electrophoretic migration velocity was significantly reduced and all were clear single bands as the number of four single-stranded DNA strands of the tetrahedral cone increased one by simple annealing, indicating the successful preparation of TDNs; on the basis of the formation of the TDNs, gradually adding annealed DNA hairpins (HMg, H1, H2 and H3) to obtain hairpin-modified functionalized TDNH, as shown in strips 5-8 of FIG. 6, the TDNH electrophoretic mobility is further slowed down along with the addition of the hairpins, which indicates that the four hairpins are successfully paired and connected to four vertexes of the TDNs through base complementation respectively; as shown in FIG. 6, lane 9, a new bright band appeared at the upper end of the electrophoresis in the presence of miRNA-21 (D), indicating the formation of the network Y-TDNH.
In order to further visually observe the network structure and the fluorescence resonance energy transfer condition of the Y-TDNH, the Y-TDNH is observed by a Transmission Electron Microscope (TEM) and a confocal microscope in the presence of an initiator. As shown in FIGS. 7A and 7B, the network structure of Y-TDNH can be clearly found from TEM photographs of Y-TDNH. FIGS. 7C and 7D are confocal images of TDNH and Y-TDNH, respectively. As can be seen from FIG. 7C, FAM fluorescence of TDNH itself is present, but fluorescence of TAMRA is almost absent; as can be seen from FIG. 7D, when miRNA-21 (D) is present, the formed Y-TDNH can undergo fluorescence resonance energy transfer.
Test example 4 in vitro FRET assay for TDNH
100nM TDNH and different concentrations of miRNA-21 (D) were incubated at 37 ℃ for 4h, with a mixture volume of 100. mu.L. Fluorescence of FAM was then excited at 488nm, fluorescence of TAMRA was further excited by fluorescence resonance energy transfer, fluorescence emission spectra of FAM and TAMRA were collected in the range of 500-650nm using a fluorescence spectrophotometer, and fluorescence of FAM at 520nm was normalized. The experiment was repeated three times.
As shown in FIG. 8A, curves a-l are fluorescence signal curves corresponding to miRNA-21 concentrations of 0pM, 5.0pM, 10pM, 50pM, 0.1 nM, 0.5 nM, 1.0 nM, 5.0 nM, 10 nM, 20nM, 50nM, 80 nM, respectively, and when the miRNA-21 (D) concentration increases, the fluorescence emission of the acceptor and donor at 585nM of TAMRA of TDNH increasesRatio (F)A/FD) The signal gradually increases; as shown in FIG. 8B, when the miRNA-21 (D) concentration is greater than 5nM, the FRET signal is slower than the rate of increase and gradually trends towards a plateau; as shown in FIG. 8C, the logarithm of the concentration of miRNA-21 (D) (0.005-5 nM) was taken to obtain the logarithm of the concentration of miRNA-21 and FA/FDThe linear equation of (a) is,y=0.226lgC+0.725, detection limit 0.74 pM.
To evaluate the selectivity of TDNH for miRNA-21, TDNH was incubated with different target RNAs (or other target DNAs) at a concentration of 100nM instead of miRNA-21 (D) at 37 ℃ for 4h and FRET intensities were compared using normalization. The experiment was repeated three times. As shown in FIG. 9, there is a certain increase in FRET signal when the three base mismatches are compared with the blank, but there is still a large difference in signal intensity when compared with miRNA-21 (D); the signals of other miRNAs are basically the same as blank signals, so the TDNH of the example 1 has good selectivity.
Test example 4 biological stability and cell Membrane penetration of TDNH
Hairpin H1, TDNH, Y-DNA and Y-TDNH solutions at a concentration of 1 μ M were mixed with DNase I solution of 0.5U/mL in equal volume, respectively, incubated at room temperature 25 ℃ for different times, and then the hydrolysis resistance of the different DNAs were compared by 1% agarose electrophoresis.
As shown in FIG. 10, under the condition of 0.5U/mL DNase I, the clear electrophoretic bands of TDNH and Y-TDNH can still be clearly observed within 6h by 1% agarose electrophoresis, and can stably exist, but the hairpin alone cannot exist. It can be concluded that TDNs can protect hairpins, avoiding degradation of hairpins.
In order to further explore the application of TDNH in-situ imaging of living cells and evaluate the biocompatibility of TDNH, HeLa cells and HEK293 cells are selected as research models, and CCK-8 detection is carried out in the presence of TDNH. HeLa cells and HEK293 cells (1X 10)4cells) were seeded into 96-well plates by trypsinization and cultured for 12h to give cells of appropriate number and density. After PBS washing twice, one part of cells are cultured for 24h by using fresh culture solution containing TDNH with different concentrations, the other part of cells are cultured for different times by using TDNH with the same concentration, then PBS washing is carried out,continuously incubating the cells for 3h by using a DMEM culture solution (100 mu L) containing 10 mu L CCK-8, finally, measuring the absorbance at 460nm of each hole by using a microplate reader in cell proliferation-toxicity detection, and calculating the cell activity.
As shown in fig. 11, TDNH has low cytotoxicity and good biocompatibility, and can be applied to in situ imaging analysis of cells.
Test example 5 fluorescence imaging of TDNH in live cells
D-A type nanomachines, which were made by grafting annealed HMg, H1-FAM, H2-TAMRA and unmodified H3 on TDNs, and D-A-A type nanomachines according to the method of example 1; D-A-A nanomachines were HMg, H1-FAM, H2-TAMRA and H3-TAMRA after annealing on TDNs.
D-A, D-A-A, D-A-D is used for in situ imaging of miRNA-21 in HeLa cells, and the specific method is that the HeLa cells are subjected to trypsinization and subculture in a 96-well plate and cultured for 12 hours. When the cells reached the appropriate number, the cells were incubated in DMEM (200. mu.L) containing three sets of TDNH (100 nM) at 37 ℃ with 5% CO2Cells were incubated for 4h under conditions and cell imaging was performed by confocal laser scanning microscopy. Fluorescence imaging uses 488nm laser to excite FAM to further excite TAMRA fluorescence through FRET, and fluorescence emission spectra of FAM and TAMRA are collected in the ranges of 490-550nm and 550-600nm respectively. As shown in FIG. 12, the fluorescent probe of D-A-D type showed strong TAMRA fluorescent signal in HeLa cells, while it showed relatively weak signals in D-A and D-A-A types.
And (4) evaluating the carrying capacity of the TDNs by taking the existence of the TDNs as a variable. As shown in FIG. 13, DAPI staining results show that the pure hairpin probe has low cellular uptake rate, seriously affects the fluorescent imaging effect, and TDNH has high cellular uptake rate, can be quickly internalized into living cells without the help of additional transfection reagents, and can be used as a network node to extend in the cytoplasm of HeLa cells to realize a network structure, so that the fluorescent imaging effect is further improved, and the intracellular fluorescent intensity is significantly higher than that of the HMg/H1/H2/H3 system without TDNs. .
Cells (MCF-7, HeLa, HepG2 and HEK 293) were passaged by trypsinization in 96-well plates and cultured for 12 h.When the cells reached the appropriate number, the cells were incubated in DMEM (200. mu.L) containing TDNH (100 nM) at 37 ℃ with 5% CO2Incubation was performed for 4h under the conditions and cell imaging was performed by confocal laser scanning microscopy. As shown in FIG. 14, the FRET signal of miRNA-21 was significant in MCF-7, HePG2 and HeLa cells, while a weak FRET signal was detected in HEK293 cells. According to the FRET signal intensity, the relative content of miRNA-21 in the four cells is MCF-7, HepG2, HeLa and HEK93 from high to low.
In conclusion, four hairpins are self-assembled on four vertexes of the TDNs, on one hand, the HMg hairpins generate a shearing amplification reaction through specific recognition of miRNA-21, and the released short chains further trigger CHA amplification; on the other hand, the spatial structure of TDNs is utilized to realize the connection of TDNH in the spatial range, the local concentration is increased, and the realization of Mg is more facilitated2+DNAzyme hairpins recognize and cleave and convert D-A-D type FRET signals. Through the shearing and CHA processes, double-cycle amplification can be realized, and more importantly, compared with the traditional single-donor single-acceptor, the FRET efficiency of the D-A-D mode is higher, so that the detection sensitivity can be further improved. Due to the programmability, special functionality and good biocompatibility of DNA, TDNH can realize detection and in-situ imaging of specific miRNA, protein and other molecules in tumor cells, and provides a new strategy for detection of cancer-related markers.
Although the present invention has been described in detail by referring to the drawings in connection with the preferred embodiments, the present invention is not limited thereto. Various equivalent modifications or substitutions can be made on the embodiments of the present invention by those skilled in the art without departing from the spirit and scope of the present invention, and these modifications or substitutions are within the scope of the present invention/any person skilled in the art can easily conceive of the changes or substitutions within the technical scope of the present invention.
SEQUENCE LISTING
<110> Qingdao university of science and technology
<120> D-A-D type FRET DNA nano machine and preparation method and application thereof
<130> 2021
<160> 17
<170> PatentIn version 3.5
<210> 1
<211> 70
<212> DNA
<213> Artificial Synthesis
<400> 1
tatcaccagg cagttgacag tgtagcaagc tgtaatagat gcgagggtcc aatactcgcg 60
attcggcaca 70
<210> 2
<211> 70
<212> DNA
<213> Artificial Synthesis
<400> 2
tcagggtcta cggcttcaac tgcctggtga taaaacgaca ctacgtggga atctactatg 60
gcggctcttc 70
<210> 3
<211> 71
<212> DNA
<213> Artificial Synthesis
<400> 3
gctcactctt ccacttttca gacttaggaa tgtgcttccc acgtagtgtc gtttgtattg 60
gaccctcgca t 71
<210> 4
<211> 70
<212> DNA
<213> Artificial Synthesis
<400> 4
tcaatgtcat cggatacatt cctaagtctg aaacattaca gcttgctaca cgagaagagc 60
cgccatagta 70
<210> 5
<211> 81
<212> DNA
<213> Artificial Synthesis
<400> 5
caaaaagaga gcaacatcag tcctragtct ttttttttga tccgagccgg acgaagggat 60
aagctatgtg ccgaatcgcg a 81
<210> 6
<211> 73
<212> DNA
<213> Artificial Synthesis
<400> 6
agccgtagac cctgagagca acatcagtcc tgtatagccc aagagagtaa ggactgatgt 60
tgctctcttt ttg 73
<210> 7
<211> 73
<212> DNA
<213> Artificial Synthesis
<400> 7
agtggaagag tgagcgtata gcccaagaga gcacctacgc aaaaagatac tctcttgggc 60
tatacaggac tga 73
<210> 8
<211> 73
<212> DNA
<213> Artificial Synthesis
<400> 8
atccgatgac attgacacct acgcaaaaag agagcaacat cagtccttat ctttttgcgt 60
aggtgctctc ttg 73
<210> 9
<211> 24
<212> DNA
<213> Artificial Synthesis
<400> 9
caaaaagaga gcaacatcag tcct 24
<210> 10
<211> 22
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<213> Artificial Synthesis
<400> 10
uagcuuauca gacugauguu ga 22
<210> 11
<211> 22
<212> DNA
<213> Artificial Synthesis
<400> 11
tagcttatca gactgatgtt ga 22
<210> 12
<211> 22
<212> DNA
<213> Artificial Synthesis
<400> 12
tagcagcacg taaatattgg cg 22
<210> 13
<211> 22
<212> DNA
<213> Artificial Synthesis
<400> 13
ttcaagtaat ccaggatagg ct 22
<210> 14
<211> 22
<212> DNA
<213> Artificial Synthesis
<400> 14
taacactgtc tggtaaagat gg 22
<210> 15
<211> 22
<212> DNA
<213> Artificial Synthesis
<400> 15
tagcttatca gactaatgtt ga 22
<210> 16
<211> 22
<212> DNA
<213> Artificial Synthesis
<400> 16
tagcttatca gactcatctt ga 22
<210> 17
<211> 22
<212> DNA
<213> Artificial Synthesis
<400> 17
tagcttttca gactcaagtt ga 22
Claims (10)
1. A preparation method of a D-A-D type FRET DNA nano machine is characterized in that four annealed hairpin DNAs are used for sequentially modifying a DNA tetrahedron to obtain the D-A-D type FRET DNA nano machine, the four hairpin DNAs are respectively HMg, H1-FAM, H2-TAMRA and H3-FAM, the sequence of the HMg is shown as SEQ ID No.5, the sequence of the H1-FAM is shown as SEQ ID No.6, the sequence of the H2-TAMRA is shown as SEQ ID No.7, and the sequence of the H3-FAM is shown as SEQ ID No. 8.
2. The method of claim 1, comprising the steps of:
(1) diluting the four hairpin DNAs with Tris-HCl buffer solutions respectively and annealing;
(2) then the four annealed hairpin DNAs are mixed with DNA tetrahedron proportion in sequence and reacted at 37 ℃, and the molar ratio of the four hairpin DNAs to the DNA tetrahedron is 1:1:1:1: 1.
3. the preparation method according to claim 2, wherein the step (1) is specifically: and (3) respectively diluting the four hairpin DNAs into 1 mu M dispersions by using Tris-HCl buffer solution, respectively heating the four dispersions at 95 ℃ for 5min, and naturally cooling to room temperature to finish annealing treatment.
4. The method of claim 3, wherein the Tris-HCl buffer solution has a concentration of 20mM and a pH of 7.4.
5. The method of claim 1, wherein the DNA tetrahedron is prepared by:
sequentially adding four long oligonucleotide chains into a TM buffer solution to obtain a uniform mixture, heating and immediately cooling to obtain a DNA tetrahedron, wherein the four long oligonucleotide chains are respectively TDNa, TDNb, TDNc and TDNd, the sequence of TDNa is shown in SEQ ID No.1, the sequence of TDNb is shown in SEQ ID No.2, the sequence of TDNc is shown in SEQ ID No.3, and the sequence of TDNd is shown in SEQ ID No. 4.
6. The method according to claim 5, wherein the mixture is heated at 95 ℃ for 5 min; the cooling temperature is 4 deg.C, and the cooling time is 30 min.
7. The method according to claim 5, wherein the concentrations of TDNa, TDNb, TDNc, and TDNd in the DNA tetrahedron are all 1 μ M.
8. The method according to claim 5, wherein the concentration of Tris-HCl in the TM buffer solution is 20mM, MgCl2Has a concentration of 50mM, and the pH of the TM buffer solution is 8.0.
9. A D-A-D type FRET DNA nanomachine prepared by the preparation method of any one of claims 1 to 8.
10. Use of a D-a-D type FRET DNA nanomachine of claim 9 for intracellular miRNA detection and/or imaging.
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